The structures of heme-based oxygenases (e.g. various cytochrome P450 (cyt P450), nitric oxide synthase (NOS) and heme oxygenase (HO)) have been long studied with the hope that such data can be used to predict product and inhibitor specificity. But most structural data has been obtained for non-productive states of the enzymes, and thus may not be relevant to the active species. In this R21, we propose the application of unusual ferrous nitroxyl adducts (HNO-FeII) of these oxygenases as structural analogues of the ferrous dioxygen adducts (O2-FeII) that directly precedes turnover. HNO is isoelectronic to O2, and is shown to bind tightly and irreversibly to O2 carriers like myoglobin, hemoglobin and leghemoglobin. The HNO-FeII adducts are diamagnetic, like the O2-FeII adducts, but much longer lived. Most importantly, the nitrosyl hydride provides a spectroscopic handle at the very center of the active pocket;the resonance in 1H NMR occurs ca. 15 ppm, well away from other protein-based signals, and therefore may be used to characterize substrate binding within the pocket in a state analogous to that proceeding turnover. The bound HNO also forms H-bonding interactions within the pocket, which are known to be important in determining oxygenase activity. Stable HNO adducts are demonstrated for two heme oxygenases, the characteristic peak ca 15 ppm seen by 1H NMR. Similar adducts have been generated in P450BM3 and P450cam with and without substrates;and similar methods are proposed to obtain such adducts of nNOS. A key proposed experiment will be to use the diamagnetic hydride resonance to map out the substrate position in P450BM3 by 2D NOE methods, as previous NMR and crystallographic studies give widely different distances. A key focus of this exploratory proposal is the development of synthetic methodologies to such adducts, following kinetics and yields of formation, assessing lifetime and possible reactivity, and determining key properties such as H/D exchange rates that would be important in their use as structural probes. Ultimately, these adducts may be of use in assessing mobility of substrate and inhibitors within the pocket. We are in a unique position to perform these studies: the PI Farmer's group first identified such an HNO adduct of myoglobin, and has characterized its structure and reactivity, as well uncovering several synthetic routes to such adducts. CoPI Poulos has longstanding interest in heme protein structure and in the characterizations of ligand and inhibitor binding;he has expertise in protein expression and crystallographic characteristics of all the target proteins (HO, P450 and NOS).Project Narrative Heme-based oxygenases are key players in the metabolism of drugs, in the synthesis of physiological effectors like nitric oxide and steroids, and in the nuetralization of toxins such as the hemes themselves. They are the key determinant of drug lifetime and action, are themselves important targets for therapeutic agents (e.g., fungal infections, cancer), are responsible for a broad range of drug toxicities. An important aspect of their activity is the ability to bind and hydroxylate targets at specific sites. The ability to predict P450 substrate and inhibitor specificity is central to all of these areas of P450 involvement.